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Fig. 2 | Journal of Hematology & Oncology

Fig. 2

From: PTENP1 is a ceRNA for PTEN: it’s CRISPR clear

Fig. 2

CRISPR-mediated knock-down of PTENP1 RNA results in the downregulation of PTEN expression and in an increase in DU145 cell proliferation. a, b Schematic representation of the location of the 4 gRNAs used to knock down PTENP1 expression (a). The sequence of PTENP1 gRNAs (#1 to #4) corresponds to the sequence of the siRNAs described in Fig. 1, extended by 3 nt (blue, b). c Luciferase assay performed in HEK293T cells to compare the efficiency of single siRNAs and gRNAs, as well as their mixes, in downregulating the expression of PTENP1. The assay was performed 36 h after the co-transfection of pGLU/ψ3′UTR plasmid with the indicated siRNAs (siCT; siPTENP1 #1,2,3,4) or gRNA-expressing plasmids (pCasRx-gRNA (SCR); pCasRx-gRNA-PTENP1 #1,2,3,4). In pGLU/ψ3′UTR plasmid, the R1 high homology and the R2 low homology regions of PTENP1 3′UTR are cloned downstream of Luciferase coding sequence [1]. d Cartoon summarizing the experimental protocol used to test the gRNAs in DU145 cells. DU145 cells were stably infected using the lentiviral vector pEF1a-CasRx-2A-eGFP, then sorted for high eGFP expression and, finally, electroporated with pCasRx-gRNA plasmid or the mix of pCasRx-gRNA-PTENP1 #1,2,3,4 plasmids. e qRT-PCR quantification of CasRx mRNA in DU145 cells that were stably infected with pEF1a-CasRx-2A-eGFP and then sorted for high eGFP expression (DU145-CasRx). f qRT-PCR quantification of the indicated transcripts 24 h after the transfection of pCasRx-gRNA (SCR) or pCasRx-gRNA-PTENP1 #1,2,3,4 mix in DU145-CasRx cells. g (left) Representative western blot detection of PTEN/GAPDH and pAKT/GAPDH proteins, performed 48 h after the transfection of pCasRx-gRNA (SCR) or pCasRx-gRNA-PTENP1 #1,2,3,4 mix in DU145-CasRx cells. (right) Quantification of protein levels. h Growth curves of DU145-CasRx cells transfected with pCasRx-gRNA (SCR) or pCasRx-gRNA-PTENP1 #1,2,3,4 mix. T1/3/5/7: days after the transfection. The results reported in fh confirm the data obtained using siRNAs: the knock-down of PTENP1 negatively affects PTEN expression. As a consequence, AKT gets hyper-phosphorylated and cell proliferation increases. The graphs represent the mean ± SEM of three independent experiments. Statistically significant differences are indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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